Crystallization of the rice immune receptor RGA5A_S with the rice blast fungus effector AVR1‐CO39 prepared <italic>via</italic> mixture and tandem strategies.

RGA5 is a component of the Pia resistance‐protein pair (RGA4/RGA5) from <italic>Oryza sativa</italic> L. <italic>japonica</italic>. It acts as an immune receptor that directly recognizes the effector AVR1‐CO39 from <italic>Magnaporthe oryzae via</italic> a C‐terminal non‐LRR domain (RGA5A_S). The interaction between RGA5A_S and AVR1‐CO39 relieves the repression of RGA4, leading to effector‐independent cell death. To determine the structure of the complex of RGA5A_S and AVR1‐CO39 and to understand the details of this interaction, the complex was prepared by fusing the proteins together, by mixing them <italic>in vitro</italic> or by co‐expressing them in one host cell. Samples purified <italic>via</italic> the first two strategies were crystallized under two different conditions. A mixture of AVR1‐CO39 and RGA5A_S (complex I) crystallized in 1.1 <italic>M</italic> ammonium tartrate dibasic, 0.1 <italic>M</italic> sodium acetate–HCl pH 4.6, while crystals of the fusion complex RGA5A_S‐TEV‐AVR1‐CO39 (complex II) were grown in 2 <italic>M</italic> NaCl. The crystal of complex I belonged to space group <italic>P</italic>3121, with unit‐cell parameters <italic>a</italic> = <italic>b</italic> = 66.2, <italic>c</italic> = 108.8 Å, α = β = 90, γ = 120°. The crystals diffracted to a Bragg spacing of 2.4 Å, and one molecule each of RGA5A_S and AVR1‐CO39 were present in the asymmetric unit of the initial model. The crystal of complex II belonged to space group <italic>I</italic>4, with unit‐cell parameters <italic>a</italic> = <italic>b</italic> = 137.4, <italic>c</italic> = 66.2 Å, α = β = γ = 90°. The crystals diffracted to a Bragg spacing of 2.72 Å, and there were two molecules of RGA5A_S and two molecules of AVR1‐CO39 in the asymmetric unit of the initial model. Further structural characterization of the interaction between RGA5A_S and AVR1‐CO39 will lead to a better understanding of the mechanism underlying effector recognition by R proteins. [ABSTRACT FROM AUTHOR]